2-Methylene-19-nor-(23S)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone and 2-methylene-19-nor-(23R)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone

ABSTRACT

Compounds of formula 1A and 1B are provided where X 1  and X 2  are independently selected from H or hydroxy protecting groups. Such compounds may be used in preparing pharmaceutical compositions and are useful in treating a variety of biological disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/666,129 filed Mar. 29, 2005, the entire contents of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to2-methylene-19-nor-(23R)-25-dehydro-1α-hydroxyvitamin D₃-26,23-lactone(“GC-3”) and 2-methylene-19-nor-(23S)-25-dehydro-1α-hydroxyvitaminD₃-26,23-lactone (“HLV”), and to pharmaceutical formulations thatinclude these compounds or mixtures thereof. The invention also relatesto the use of GC-3, HLV, salts thereof, and mixtures thereof in thepreparation of medicaments for use in treating various diseases.

BACKGROUND OF THE INVENTION

The natural hormone, 1α,25-dihydroxyvitamin D₃ (also referred to as1α,25-dihydroxycholecalciferol and calcitriol) and its analog in theergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins, and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.The structure of 1α, 25-dihydroxyvitamin D₃ and the numbering systemused to denote the carbon atoms in this compound are shown below.

1α,25-Dihydroxyvitamin D₃=1α,25-Dihydroxycholecalciferol=Calcitriol

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by theChugai group as potential drugs for osteoporosis and as antitumoragents. See also Okano et al., Biochem. Biophys. Res. Commun. 163, 1444(1989). Other 2-substituted (with hydroxyalkyl, e.g., ED-120, andfluoroalkyl groups) A-ring analogs of 1α, 25-dihydroxyvitamin D₃ havealso been prepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41,1111 (1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993);Posner et al., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617(1995)).

Various 2-substituted analogs of 1α, 25-dihydroxy-19-nor-vitamin D₃ havealso been synthesized, i.e. compounds substituted at the 2-position withhydroxy or alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with2-alkyl groups (DeLuca et al., U.S. Pat. No. 5,945,410), and with2-alkylidene groups (DeLuca et al., U.S. Pat. No. 5,843,928), whichexhibit interesting and selective activity profiles. All these studiesindicate that binding sites in vitamin D receptors can accommodatedifferent substituents at C-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-Hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while1α-hydroxy-2-methylene-19-nor-(20S)-homopregnacalciferol is described inU.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to the vitamin D receptor and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

SUMMARY OF THE INVENTION

The invention provides2-methylene-19-nor-(23R)-25-dehydro-1α-hydroxyvitamin D₃-26,23-lactone(“GC-3”), 2-methylene-19-nor-(23S)-25-dehydro-1α-hydroxyvitaminD₃-26,23-lactone (“HLV”), and related compounds, pharmaceuticalformulations that include GC-3 and/or HLV, and the use of thesecompounds or mixtures thereof in the preparation of medicaments for usein treating various disease states.

Therefore, in one aspect, the invention provides a vitamin D analog thatincludes a lactone functional group. In some such embodiments, thelactone includes an exocyclic methylene group. In some such embodiments,the exocyclic methylene group is bonded to the carbon atom adjacent tothe carbonyl moiety of the lactone functional group. In someembodiments, the vitamin D analog is a 19-nor vitamin D analog.

Therefore, in one aspect, the invention provides compounds having theformula 1A, formula 1B, or a mixture thereof as shown below:

where X¹ and X² may be the same or different and are independentlyselected from H or hydroxy-protecting groups. In some embodiments, X¹and X² are both hydroxy protecting groups such as silyl groups. In somesuch embodiments, X¹ and X² are both t-butyldimethylsilyl groups. Inother embodiments, X¹ and X² are both H such that the compound shown asformula 1A above is2-methylene-19-nor-(23R)-25-dehydro-1α-hydroxyvitamin D₃-26,23-lactoneand the compound shown as formula 1B is2-methylene-19-nor-(23S)-25-dehydro-1α-hydroxyvitamin D₃-26,23-lactone,or mixtures thereof, having the isomeric formulas 1A1 and 1B1 as shownbelow:

In some such embodiments, the compounds of formulas 1A1 and 1B1 arecompounds of formulas 1A2 and 1B2, or mixtures thereof, and have thestructures shown below:

In some such embodiments, the compounds may be present in a purifiedform. In other embodiments, the compounds in a composition may bepresent as a mixture. In some embodiments, the mixture includes thecompound of formula 1A and the compound of formula 1B, and the ratio ofthe compound of formula 1A to the compound of formula 1B ranges from50:50 to 99.9:0.1. In some such embodiments, the ratio of the compoundof formula 1A to the compound of formula 1B ranges from 70:30 to99.9:0.1, from 80:20 to 99.9:0.1, from 90:10 to 99.9:0.1, or from 95:5to 99.9:0.1. In other embodiments, the mixture includes the compound offormula 1A and the compound of formula 1B, and the ratio of the compoundof formula 1B to the compound of formula 1A ranges from 50:50 to99.9:0.1. In some such embodiments, the ratio of the compound of formula1B to the compound of formula 1A ranges from 70:30 to 99.9:0.1, from80:20 to 99.9:0.1, from 90:10 to 99.9:0.1, or from 95:5 to 99.9:0.1.

The above compounds exhibit desired, and highly advantageous, patternsof biological activity. Both GC-3 and HLV bind to the vitamin Dreceptor, but both of these compounds are less active in this respectthan is 1α, 25-dihydroxyvitamin D₃. Both GC-3 and HLV also show lessactivity than 1,25-(OH)₂D₃ in inducing differentiation of HL-60 cells.However, GC-3 and HLV have the ability to antagonize1α,25-dihydroxyvitamin D₃-mediated transcription. GC-3 and HLV have nocalcemic activity when measured by bone calcium mobilization, but doretain the ability to elevate intestinal calcium transport. Becausethese compounds act as antagonists in vitro and weak agonists in vivo,these compounds could serve as useful therapeutic agents whenadministered locally to some tissues. These compounds may thus find usein therapies for treating asthma, hypercalcemia, eczema, sarcoidosis,and vitamin D intoxication. These compounds are characterized byrelatively high binding to the vitamin D receptor compared to1α,25-dihydroxyvitamin D₃, while also retaining the ability to elevateintestinal calcium transport. However, these compounds appear to have nocalcemic activity as compared to 1α,25-dihydroxyvitamin D₃ in theirability to mobilize calcium from bone. These compounds also showantagonistic activity when administered along with1α,25-dihydroxyvitamin D₃. Thus, these compounds may be useful intherapies for treating asthma, hypercalcemia, eczema, sarcoidosis, andvitamin D intoxication.

The compounds described herein are also characterized by moderate celldifferentiation activity. Thus, these compounds may also be used astherapeutic agents for the treatment of psoriasis and/or an anti-canceragents, especially against leukemia, colon cancer, breast cancer andprostate cancer. In addition, due to their moderate cell differentiationactivities, the compounds may be used as therapeutic agents for thetreatment of various skin conditions including wrinkles, lack ofadequate dermal hydration, i.e. dry skin, lack of adequate skinfirmness, i.e. slack skin, and insufficient sebum secretion. Use of thecompounds thus moisturizes skin and improves the barrier function ofskin.

In another aspect, the invention provides a method of antagonizing thevitamin D receptor. The method includes administering a compound orpharmaceutical composition of the invention to an animal subject. Thecompound administered to the subject antagonizes the vitamin D receptor.

In another aspect, the invention provides a method of treating asthma oreczema in an animal subject suffering from asthma or eczema. The methodincludes administering an effective amount of a compound or apharmaceutical composition of the invention to the animal subject.Administration of the compound leads to a reduction in the symptomsassociated with asthma or eczema.

In some embodiments of the methods of the invention, the compound orpharmaceutical composition is administered orally, rectally,parenterally, transdermally, or topically. In other embodiments, thecompound or pharmaceutical formulations is administered in an aerosolwhich may be accomplished using an inhaler or a nebulizer.

In another aspect, the invention provides the use of a compound of theinvention in the preparation of a pharmaceutical composition ormedicament for antagonizing the vitamin D receptor and/or for treatingasthma or eczema in an animal subject suffering from asthma or eczema.In some embodiments, the compounds are used to prepare an aerosol whichmay include a glycol compound such as propylene glycol.

The compounds of the invention may be used to prepare pharmaceuticalformulations or medicaments that include a compound or a mixture of thecompounds of the invention in combination with a pharmaceuticallyacceptable carrier. Such pharmaceutical formulations and medicaments maybe used to treat various biological disorders such as those describedherein, including those mediated by a vitamin D receptor. Methods fortreating such disorders typically include administering an effectiveamount of the compound, or an appropriate amount of a pharmaceuticalformulation or a medicament that includes the compound, to a subjectsuffering from the biological disorder. In some embodiments, the subjectis a mammal. In some such embodiments, the mammal is selected from arodent, a primate, a bovine, an equine, a canine, a feline, an ursine, aporcine, a rabbit, or a guinea pig. In some such embodiments, the mammalis a rat or is a mouse. In some embodiments, the subject is a primatesuch as, in some embodiments, a human.

The compounds may be present in a composition to treat the above-noteddiseases and disorders in an amount from about 0.01 μg/gm to about 1mg/gm of the composition, preferably from about 0.1 μg/gm to about 500μg/gm of the composition, and may be administered topically,transdermally, orally, rectally, or parenterally in dosages of fromabout 0.01 μg/day to about 1 mg/day, preferably from about 0.1 μg/day toabout 500 μg/day.

Further objects, features and advantages of the invention will beapparent from the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 illustrate various biological activities of2-methylene-19-nor-(23R)-1α,25-dihydroxyvitamin D₂ (referred to as“GC-3” in the Figures) and2-methylene-19-nor-(23S)-1α,25-dihydroxyvitamin D₂ (referred to as “HLV”in the Figures) compared with those of the native hormone1α,25-dihydroxyvitamin D₃ (referred to as “1,25(OH)₂D₃” in the Figures).

FIG. 1 is a graph comparing the relative activity of GC-3, HLV, and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor.

FIG. 2 is a graph comparing the percent HL-60 cell differentiation as afunction of concentration of GC-3, HLV, and 1,25(OH)₂D₃.

FIG. 3 is a graph comparing the in vitro transcription activity of1,25(OH)₂D₃ alone with that of 1,25(OH)₂D₃ in combination with GC-3.

FIG. 4 is a graph comparing the in vitro transcription activity of1,25(OH)₂D₃ alone with that of 1,25(OH)₂D₃ in combination with HLV.

FIG. 5 is a graph comparing the in vitro transcription activity of HLV,GC-3, and 1,25(OH)₂D₃.

FIG. 6 is a bar graph comparing the bone calcium mobilization activityof GC-3, HLV, and 1,25(OH)₂D₃.

FIG. 7 is a bar graph comparing the intestinal calcium transportactivity of GC-3, HLV, and 1,25(OH)₂D₃.

DETAILED DESCRIPTION OF THE INVENTION

2-Methylene-19-nor-(23R)-25-dehydro-1α-hydroxyvitamin D₃-26,23-lactone(“GC-3”) and 2-methylene-19-nor-(23S)-25-dehydro-1α-hydroxyvitaminD₃-26,23-lactone (“HLV”) were synthesized, and tested, and found to beuseful in treating a variety of biological disorders as describedherein. Structurally, GC-3 has the formula 1A1 and HLV has the formula1B1, as shown below:

One step in the reaction sequence used in the preparation of the GC-3and HLV isomers can be accomplished by condensing an appropriatebicyclic Windaus-Grundmann type ketone (II) with the allylic phosphineoxide III followed by TES removal, side chain elongation, lactone ringformation, and deprotection (removal of the Y₁ and Y₂ groups), in alater step.

In phosphine oxide III, Y₁ and Y₂ are preferably hydroxy-protectinggroups such as silyl protecting groups. The t-butyldimethylsilyl (TBDMS)group is an example of a particularly useful hydroxy-protecting group.The process described above represents an application of the convergentsynthesis concept, which has been applied effectively to the preparationof numerous vitamin D compounds (see Lythgoe et al., J. Chem. Soc.Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Tohet al., J. Org. Chem. 48, 1414 (1983); Baggiolini et al., J. Org. Chem.51, 3098 (1986); Sardina et al., J. Org. Chem. 51, 1264 (1986); J. Org.Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca etal., U.S. Pat. No. 5,536,713; and DeLuca et al., U.S. Pat. No. 5,843,928all of which are hereby incorporated by reference in their entirety andfor all purposes as if fully set forth herein).

Phosphine oxide III is a convenient reagent that can be used to preparea large number of 19-nor vitamin D compounds and may be preparedaccording to the procedures described by Sicinski et al., J. Med. Chem.,41, 4662 (1998), DeLuca et al., U.S. Pat. No. 5,843,928; Perlman et al.,Tetrahedron Lett. 32, 7663 (1991); and DeLuca et al., U.S. Pat. No.5,086,191. Scheme I shows the general procedure for synthesizingphosphine oxide III as outlined in U.S. Pat. No. 5,843,928 which ishereby incorporated by reference in its entirety as if fully set forthherein. Modification of the method shown in Scheme I may be used toproduce a large number of vitamin D analogs as will be apparent to thoseskilled in the art. For example, a wide variety of phosphonium compoundsmay be used in place of the MePh₃P⁺Br⁻ used to convert ketone B toalkene C. Examples of such compounds include EtPh₃P⁺Br⁻, PrPh₃P⁺Br⁻, andcompounds generally prepared by reaction of triphenylphosphine with analkyl halide, an alkenyl halide, a protected-hydroxyalkyl halide, and aprotected hydroxyalkenyl halide. Alkenes prepared using this proceduremay then be carried through to prepare a phosphine oxide in an analogousmanner to that used to prepare phosphine oxide H in Scheme I.Alternatively, an alkene analogous to compound C of Scheme I may bereduced with (Ph₃P)₃RhCl and H₂ to provide other vitamin D analogs. SeeU.S. Pat. No. 5,945,410 and Sicinski, R. R. et al., J. Med. Chem., 41,46624674 (1998) both of which are hereby incorporated by reference intheir entireties and for all purposes. Therefore, the procedure forforming the phosphine oxide shown in Scheme I may be used to prepare awide variety of vitamin D analogs in addition to the compound of thepresent invention.

Hydroindanones of structure II can prepared by known methods or adaptedmethods as will be readily apparent to one of skill in the art anddescribed herein. Specific examples of some important bicyclic ketonesused to synthesize vitamin D analogs are those described in Mincione etal., Synth. Commun 19, 723, (1989); and Peterson et al., J. Org. Chem.51, 1948, (1986).

An overall process for synthesizing 2-alkylidene-19-nor-vitamin Dcompounds is illustrated and described in U.S. Pat. No. 5,843,928 whichis hereby incorporated by reference in its entirety and for all purposesas if fully set forth herein.

As used herein, the term “hydroxy-protecting group” signifies any groupcommonly used for the temporary protection of the hydroxy (—OH)functional group, such as, but not limited to, alkoxycarbonyl, acyl,alkylsilyl or alkylarylsilyl groups (hereinafter referred to simply as“silyl” groups), and alkoxyalkyl groups. Alkoxycarbonyl protectinggroups are alkyl-O—CO— groups such as methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The term“acyl” signifies an alkanoyl group of 1 to 6 carbons, in all of itsisomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as anoxalyl, malonyl, succinyl, glutaryl group, or an aromatic acyl groupsuch as benzoyl, or a halo, nitro or alkyl substituted benzoyl group.Alkoxyalkyl protecting groups are groups such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyidimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group. An extensive listof protecting groups for the hydroxy functionality may be found inProtective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G. M.,John Wiley & Sons, New York, N.Y., (3rd Edition, 1999) which can beadded or removed using the procedures set forth therein and which ishereby incorporated by reference in its entirety and for all purposes asif fully set forth herein.

A “protected hydroxy” group is a hydroxy group derivatized or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functional groups, e.g., the silyl, alkoxyalkyl,acyl or alkoxycarbonyl groups, as previously defined.

EXAMPLES Synthesis of2-methylene-19-nor-(23R)-25-dehydro-1α-hydroxyvitamin D₃-26,23-lactone(GC-3) and 2-methylene-19-nor-(23S)-25-dehydro-1α-hydroxyvitaminD₃-26,23-lactone (HLV)

The synthesis and characteristics of various 19-nor vitamin D analogs isdescribed in numerous United States patents including U.S. Pat. No.5,843,928, U.S. Pat. No. 6,627,622, U.S. Pat. No. 6,579,861, U.S. Pat.No. 5,086,191, U.S. Pat. No. 5,585,369, and U.S. Pat. No. 6,537,981.Each of the above-described references is hereby incorporated byreference in its entirety and for all purposes as if fully set forthherein.

Schemes I, IIA, IIB, and IIC outline the synthetic procedures describedbelow, in detail.

8S,20S)-de-A,B-8-hydroxy-20-(hydroxymethyl)-pregnane (1)

A flame-dried 1000 mL three-necked flask was charged sequentially with5.0 g (12.7 mmol) of ergocalciferol (commercially available fromSigma-Aldrich), 400 mL of anhydrous MeOH, and 5 mL (62 mmol) ofanhydrous pyridine. The solution was cooled at −78° C. and treated withO₃ until a deep blue color developed and persisted (approximately 20-30minutes). The solution was subsequently flushed with O₂ for 15 minutesuntil the blue color faded. Solid sodium borohydride (4.5 g, 118.9 mmol)was added in portions over a 4 hour time period. The first portion ofsodium borohydride was added at −78° C., after 20 minutes a secondportion of sodium borohydride was added. The reaction mixture was warmedto room temperature over a period of 3-4 hours, then the last portion ofsodium borohydride was added. After being stirred for 18 hours at roomtemperature, the mixture was quenched with water, concentrated in vacuoand extracted with EtOAc. The combined organic phases were washed with 1N HCl, saturated NaHCO₃, brine, dried over MgSO₄, filtered, andconcentrated in vacuo. Purification by silica gel chromatography (15%EtOAc/hexanes) afforded 1.41 g (6.64 mmol) of diol in 52% yield as awhite solid; ¹H NMR (CDCl₃, 600 MHz) δ 4.09 (dm, J=3.0 Hz, 1H), 3.64(dd, J=10.5, 3.0 Hz, 1H), 3.38 (dd, J=10.5, 6.6 Hz, 1H) 1.99 (dm, J=13.2Hz, 1H), 1.03 (d, J=7.2 Hz, 3H), 0.96 (s, 3H); ¹³C NMR (CDCl₃) δ 69.26,67.87, 52.97, 52.40, 41.89, 40.26, 38.27, 33.60, 26.69, 22.60, 17.43,16.65, 13.61; exact mass calculated for C₁₃H₂₄O₂ [M]⁺ 212.1776, found212.1779.

(8S,20R)-de-A,B-8-hydroxy-20-(formylmethyl)-pregnane (3)

A solution of diol 1 (500 mg, 2.35 mmol) in 5 mL of anhydrous pyridinewas cooled to −25° C. A precooled solution of tosyl chloride (553 mg,2.9 mmol) in 1 mL of anhydrous pyridine was added dropwise to the diolsolution via cannula. Upon stirring for 3.5 hours at −25° C., thereaction was warmed up to 0° C. and allowed to stir for an additional 20hours. The mixture was extracted with CH₂Cl₂, washed with saturatedCuSO₄ aqueous solution, dried over MgSO₄, filtered, and concentrated togive a residue which was chromatographed on a silica gel column (20%EtOAc/hexanes) to afford 600 mg (1.64 mmol) of the correspondingtosylate 2 in 70% yield. To a solution of 2 (300 mg, 0.82 mmol) in DMSO(2 mL) was added KCN (106 mg, 1.64 mmol), and the mixture was stirred at70° C. for 1.5 hours. The mixture was diluted with Et₂O and the organiclayer was washed with H₂O and saturated NaCl aqueous solution, driedover MgSO₄ and concentrated. The residue was dissolved in CH₂Cl₂ (3 mL).To the solution was added a solution of DIBALH in toluene (0.9 mL, 0.902mmol) at 0° C., and the mixture was stirred at the same temperature for1.5 hours. To the mixture was added, 10% potassium sodium tartrateaqueous solution, and the aqueous layer was extracted with Et₂O. Theorganic layer was washed with saturated aqueous NaCl solution, driedover MgSO₄ and concentrated. The residue was purified by flash columnchromatography on silica gel (3% EtOAc/hexanes) to give 3 (133 mg, 0.59mmol in 2 steps, 72%): [α]²⁰ _(D)+18.80 (c 1.21, CHCl₃); ¹H NMR (CDCl₃,400 MHz) δ 9.75 (d, J=2.4 Hz, 1H), 4.08 (s, 1H), 2.45 (dm, J=15.7 Hz,1H), 2.15 (m, 1H), 1.00 (d, J=6.6 Hz, 3H), 0.98 (s, 3H); ¹³C NMR(CDCl₃,400 MHz) δ 69.17, 56.34, 52.54, 50.68, 41.99, 40.22, 33.54,31.22, 27.40, 22.44, 19.85, 17.34, 13.50; exact mass calculated forC₁₄H₂₄O₂ [M]⁺ 224.1776, found 224.1771.

(8S,20R)-de-A,B-8-hydroxy-20-(2-hydroxyethyl)-pregnane (4)

To a solution of 3 (100 mg, 0.443 mmol) in anhydrous EtOH (10 mL) wasadded NaBH₄(85 mg, 2.22 mmol) at 0° C., and the mixture was stirred atthe same temperature for 1.5 hours. The mixture was diluted with EtOAc,washed with water, 1 N HCl, saturated aqueous NaHCO₃ solution, brine,dried over MgSO₄, and concentrated. The residue was purified by flashcolumn chromatography on silica gel (10% EtOAc/hexanes) to give thedesired diol 4 (75 mg, 0.337 mmol, 76%) ¹H NMR (CDCl₃, 800 MHz) δ 4.06(d, J=2.4 Hz, 1H), 3.69 (ddd, J=10.4, 8.8, 4.8 Hz 1H), 3.62 (ddd,J=10.4, 7.2, 7.2 Hz, 1H) 1.98 (dm, J=12.8 Hz, 1H), 0.92 (d, J=6.6 Hz,3H), 0.91 (s, 3H) ¹³C NMR (CDCl₃) δ 69.55, 61.05, 57.11, 52.82, 42.16,40.61, 39.00, 33.78, 32.67, 27.49, 22.73, 18.93, 17.64, 13.70; exactmass calculated for C₁₄H₂₆O₂ [M]⁺ 226.1933, found 226.1945.

(8S,20R)-de-A,B-8-hydroxy-20-(2-triethylsilyloxyethyl)-pregnane (5)

To a solution of diol 4 (50 mg, 0.22 mmol) in anhydrous CH₂Cl₂ (5 mL) at0° C. was added triethylamine (95 μL, 0.67 mmol) followed bytriethylsilylchloride (TESCI, 40 μL, 0.22 mmol). The solution wasstirred at 0° C. for 30 minutes and then quenched with water. Themixture was extracted with CH₂Cl₂, and the combined organic phases weredried over MgSO₄, filtered, and concentrated to give a residue that waschromatographed on silica gel column (30% EtOAc/hexanes) to afford theO-silylated compound 5 (68 mg, 0.18 mmol, 81%): [α]²⁰ _(D)+31.20 (c1.45, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 4.08 (s, 1H), 3.68 (m, 1H), 3.59(m, 1H), 1.99 (m, 1H), 0.97 (t, J=7.9 Hz, 9H), 0.91 (s, 3H), 0.60 (q,J=7.9 Hz, 6H); ¹³C NMR (CDCl₃) δ 69.45, 60.95, 56.84, 52.60, 41.89,40.35, 38.86, 33.53, 32.47, 27.19, 22.52, 18.91, 17.41, 13.41, 6.56,5.78; exact mass calculated for C₂₀H₄₀O₂Si [M]⁺ 340.2798, found340.2803.

(20R)-de-A,B-20-(2-triethylsilyloxyethyl)-pregnan-8-one (6)

To a solution of alcohol 5 (41 mg, 0.12 mmol) in CH₂Cl₂ (10 mL) wereadded pyridinium p-toluene sulfonate (PPTS) (10 mg, 0.02 mmol) andpyridinium dichromate (PDC, 0.231 g, 0.60 mmol) at room temperature.After being stirred 6 hours at room temperature, the mixture was passedthrough 2 cm of flash silica gel pad and washed with EtOAc. The filtratewas concentrated and chromatographed with 20% EtOAc in hexanes to givedesired ketone 6 (31 mg, 0.091 mmol, 76%) as a colorless oil. Foranalytical purpose a sample of ketone 6 was further purified by HPLC(Zorbax Silica, 250/9.4 mm, 10% EtOAc/hexane, 5 mL/minute, Rv=23 mL); ¹HNMR (CDCl₃, 600 MHz) δ 3.67 (ddd J=10.2, 8.4, 4.8 Hz, 1H), 3.59 (ddd,J=10.2, 7.8, 7.2 Hz, 1H), 2.43 (dd, J=12.0, 7.2 Hz, 1H), 0.95 (d, J=6.6Hz, 3H) 0.94 (t, J=7.8 Hz, 9H) 0.62 (s, 3H), 0.57 (q, J=7.8 Hz, 6H), ¹³CNMR (CDCl₃) δ 212.09, 61.99, 60.72, 56.89, 49.94, 40.97, 38.95, 38.79,32.64, 27.51, 24.04, 19.08, 19.03, 12.41, 6.80, 4.42; exact masscalculated for C₁₈H₃₃O₂Si [M-C₂H5]⁺ 309.2250, found 309.2252.

(7E)-(1R,3R,20R)-1,3-di-(tert-butyldimethylsilyloxy)-2-methylene-9,10-seco-19-nor-20-(2-triethylsilyloxyethyl)-5,7-pregnandiene(7)

To a solution of phosphine oxide H (89 mg, 0.15 mmol) in anhydrous THF(600 μL) at −20° C. was slowly added PhLi (130 μL, 0.15 mmol) underargon with stirring. The solution turned deep orange. The mixture wascooled to −78° C., and a precooled (−78° C.) solution of ketone 6 (29mg, 86 μmol) in anhydrous THF (400 μL) was slowly added. The mixture wasstirred under argon at −78° C. for 1 hour and at 0° C. for 18 hours.Ethyl acetate was added, and the organic phase was washed with brine,dried (MgSO₄), and evaporated. The residue was dissolved in hexane,applied on a silica Sep-Pack cartridge, and washed with 0.5% EtOAc inhexanes to give the 19-norvitamin derivative 7 (48 mg, 79%). TheSep-Pack was then washed with ethyl acetate to recover diphenylphosphineoxide (20 mg). ¹H NMR (CDCl₃, 900 MHz) δ 6.21 (d, J=11.2 Hz, 1H, 6-H),5.83 (d, J=11.2 Hz, 1H, 7-H), 4.97 (s, 1H, =CH₂), 4.92 (s, 1H, =CH₂),4.42 (m, 2H, 1β- and 3α-H), 3.68 (ddd, J=10.4, 8.1, 4.5 Hz, 1H), 3.59(ddd, J=10.4, 7.2, 7.2 Hz, 1H), 2.82 (br d, J=12.6 Hz, 1H), 2.51 (dd,J=13.5, 5.4 Hz, 1H), 2.46 (dd, J=12.6, 4.5 Hz, 1H), 2.33 (dd, J=13.5,3.6 Hz, 1H), 2.18 (dd, J=11.7, 8.1 Hz, 1H) 0.96 (t, J=8.1 Hz, 9H,SiCH₂CH₃) 0.94 (d, J=6.3 Hz, 3H), 0.89 (s, 9H, Si-t-Bu), 0.86 (s, 9H,Si-t-Bu), 0.60 (q, J=8.1 Hz, 6H, SiCH₂), 0.54 (s, 3H, 18-H₃), 0.08,0.06, 0.04, and 0.02 (each s, each 3H, 4× SiCH₃); ¹³C NMR (CDCl₃) δ153.22, 141.37, 132.98, 122.63, 116.37, 106.48, 72.73, 71.88, 61.24,56.96, 56.52, 47.83, 45.95, 40.80, 39.27, 38.80, 33.50, 28.97, 27.93,26.06, 26.00, 23.65, 22.47, 19.47, 18.47, 18.39, 12.22, 7.03, −4.63 (2×SiMe), −4.68 (SiMe), −4.87 (SiMe); exact mass calculated for C₄₁H₇₈O₃Si₃[M]⁺ 702.5259, found 702.5286.

(7E)-(1R,3R,20R)-1,3-di-(tert-butyidimethylsilyloxy)-2-methylene-9,10-seco-19-nor-20-(2-hydroxyethyl)-5,7-pregnandiene(8)

To a solution of 7 (48 mg, 68 μmol) in benzene (2 mL) was addedAcOH/THF/H₂O (8:8:1, 8 mL) at 0° C., and was stirred at the sametemperature for 3 hours. To the mixture was added saturated aqueousNaHCO₃ solution, and the aqueous layer was extracted with EtOAc. Theorganic layer was washed with saturated aqueous NaCl solution, driedover MgSO₄, and concentrated. The residue was chromatographed on silicacolumn (5% EtOAc/hexanes) to give the desired alcohol 8. (32 mg, 54μmol, 80%); [α]²⁰ _(D)+29.2° (c 1.11, CHCl₃) ¹H NMR (CDCl₃, 800 MHz) δ6.19 (d, J=11.2 Hz, 1H, 6-H), 5.82 (d, J=11.2 Hz, 1H, 7-H), 4.95 (s, 1H,=CH₂), 4.90 (s, 1H, =CH₂), 4.40 (m, 2H, 1β- and 3α-H), 3.71 (m, 1H),3.63 (m, 1H), 2.82 (br d, J=12.8 Hz, 1H), 2.50 (dd, J=13.6, 6.4 Hz, 1H),2.44 (dd, J=12.8, 4.0 Hz, 1H), 2.30 (dm, J=10.4, Hz, 1H), 2.16 (dd,J=12.8, 8.8, 1H), 0.95 (d, J=7.2 Hz, 3H), 0.88 (s, 9H, Si-t-Bu), 0.84(s, 9H, Si-t-Bu), 0.54 (s, 3H), 0.059, 0.044, 0.028, and 0.003 (each s,each 3H, 4× SiCH₃), ¹³C NMR (CDCl₃) δ 141.01, 132.85, 125.51, 122.37,116.21, 106.26, 72.54, 71.61, 60.93, 56.78, 56.27, 47.62, 45.72, 40.59,38.95, 38.55, 33.25, 28.72, 27.81, 25.84, 25.78, 23.40, 22.22, 18.99,18.25, 18.16, 12.04, 4.86 (2× SiMe), −4.91 (SiMe), −5.10 (SiMe); exactmass calculated for C₃₅H₆₅O₃Si₂ [MH]⁺ 589.4472, found 589.4472.

(7E)-(1R,3R,20R)-1,3-di-(tert-butyidimethylsilyloxy)-2-methylene-9,10-seco-19-nor-20-(2-formylmethyl)-5,7-pregnandiene(9)

To a solution of DMSO (100 μL, 1.35 mmol) in CH₂Cl₂ (5 mL) at −60° C.oxalyl chloride (65 μL, 0.71 mmol) was added. After 2 minutes, a −60° C.solution of the primary alcohol 8 (32 mg, 55 μmol) in CH₂Cl₂ (3 mL) wasadded via cannula. The resulting mixture was stirred at −60° C. for 1hour, quenched with Et₃N (0.400 mL, 2.82 mmol), and warmed up to roomtemperature. Upon dilution with H₂O, the extraction mixture wasextracted with CH₂Cl₂, dried (MgSO₄), filtered, concentrated, andpurified by flash column chromatography (2-5% EtOAc/hexanes) to give thedesired aldehyde 9 (25 mg, 0.55 mmol, 78%) [α]²⁰ _(D) −8.4° (c 1.25,CHCl₃); ¹H NMR (CDCl₃, 600 MHz) δ 9.75 (dd, J=3.6, 1.8 Hz, 1H), 6.20 (d,J=11.4 Hz, 1H, 6-H), 5.83 (d, J=11.4 Hz, 1H, 7-H), 4.96 (s, 1H, =CH₂),4.91 (s, 1H, =CH₂), 4.41 (m, 2H, 1β- and 3α-H), 2.81 (dd, J=12.6, 4.2Hz, 1H), 2.50 (dd, J=13.2, 6.0 Hz, 1H), 2.47 (m, 2H), 2.45 (dd, J=12.6,4.8 Hz, 1H), 2.31 (dd, J=13.2, 3.0 Hz, 1H), 1.02 (d, J=6.6 Hz, 3H), 0.88(s, 9H, Si-t-Bu), 0.85 (s, 9H, Si-t-Bu), 0.58 (s, 3H), 0.07, 0.05, 0.03,and 0.01 (each s, each 3H, 4× SiCH₃); ¹³C NMR (CDCl₃) δ 203.58, 153.13,140.80, 133.32, 122.50116.61, 106.54, 72.74, 71.81, 56.45, 51.06, 47.84,45.94, 40.64, 38.78, 32.17, 32.04, 28.86, 28.14, 26.05, 26.00, 23, 54,22.38, 20.35, 18.47, 18.39, 12.29, −4.63 (3× SiMe), −4.89 (SiMe); exactmass calculated for C₃₅H₆₂O₃Si₂ [M]⁺ 586.4238, found 586.4247.

(7E)-(1R,3R,20R)-1,3-di-(tert-butyldimethylsilyloxy)-2-methylene-9,10-seco-19-nor-20-[(2S)-hydroxy-4-methoxycarbonyl-4-penten-1-yl]-5,7-pregnandiene(10) and(7E)-(1R,3R,20R)-1,3-di-(tert-butyidimethylsilyloxy)-2-methylene-9,10-seco-19-nor-20-[(2R)-hydroxy4-methoxycarbonyl-4-penten-1-yl]-5,7-pregnandiene (1)

To a solution of 9 (25 mg, 425 μmol) in saturated aqueous NH₄Clsolution/THF (5:1, 3 mL) were added methylbromomethylacrylate (10 μL, 85μmol) (commercially available from Sigma-Aldrich) and activated Zn dust(11 mg, 0.17 mmol) at 0° C., and the mixture was stirred at the sametemperature for 1.5 hours. The mixture was diluted with EtOAc. Theorganic layer was washed with saturated aqueous NaCl solution, driedover MgSO₄, and concentrated. The residue was purified by flash columnchromatography on silica gel (5% EtOAc/hexanes) to give 10 (5 mg, 7.3μmol, 57%) and 11 (4 mg, 5.5 μmol, 43%) as colorless oils, respectively.

10: ¹H NMR (CDCl₃, 400 MHz) δ 6.25(s, 1H, =CH₂), 6.21 (d, J=11.2 Hz, 1H,6-H), 5.84 (d, J=11.2 Hz, 1H, 7-H), 5.67 (s, 1H, =CH₂), 4.97 (s, 1H,=CH₂), 4.91 (s, 1H, =CH₂), 4.42 (m, 2H, 1β- and 3α-H), 3.87 (m, 1H,CH—OH), 3.77 (s, 3H, —CO₂CH₃), 2.82 (br d, J=11.3, 1H), 2.56-2.44 (m,3H), 2.40-2.25 (m, 2H), 2.18 (m, 1H), 0.97 (d, J=6.6 Hz, 3H), 0.90 (s,9H, Si-t-Bu), 0.85 (s, 9H, Si-t-Bu), 0.57 (s, 3H), 0.08, 0.06, 0.05, and0.02 (each s, each 3H, 4× SiCH₃); ¹³C NMR (CDCl₃) δ 168.31, 153.19,141.28, 137.76, 133.03, 127.92, 122.58, 116.41, 106.44, 72.79, 71.78,67.85, 57.35, 56.54, 52.28, 47.85, 46.00, 43.93, 41.78, 40.86, 38.72,33.09, 28.94, 28.08, 26.04, 25.97, 23.62, 22.44, 18.87, 18.45, 18.35,12.35, −4.67 (SiMe), 4.90 (3× SiMe); exact mass calculated forC₄₀H₇₀O₅Si₂Na [M+Na]⁺ 709.4660, found 709.4680.

11: ¹H NMR (CDCl₃, 400 MHz) δ 6.29 (s, 1H, =CH₂), 6.22 (d, J=11.1 Hz,1H, 6-H), 5.84 (d, J=11.1 Hz, 1H, 7-H), 5.70 (s, 1H, =CH₂), 4.98 (s, 1H,=CH₂), 4.93 (s, 1H, =CH₂), 4.43 (m, 2H, 1β- and 3α-H), 3.90 (m, 1H,CH—OH), 3.79 (s, 3H, —CO₂CH₃), 2.84 (br d, J=12.1 Hz, 1H), 2.71 (dd,J=13.8, 1.7 Hz, 1H), 2.56-2.42 (m, 2H), 2.34 (m, 1H), 1.03 (d, J=6.4 Hz,3H), 0.90 (s, 9H, Si-t-Bu), 0.87 (s, 9H, Si-t-Bu), 0.57 (s, 3H), 0.09,0.08, 0.06, and 0.04 (each s, each 3H, 4× SiCH₃); ¹³C NMR (CDCl₃), δ168.30, 153.17, 141.23, 137.77, 133.04, 128.07, 122.58, 116.41, 106.46,72.72, 71.83, 69.45, 57.47, 56.43, 52.28, 47.80, 45.91, 44.13, 40.81,40.27, 38.77, 34.39, 28.93, 28.12, 26.03, 25.98, 23.61, 22.42, 19.55,18.74, 18.37, 12.29, −4.67 (3× SiMe), −4.88 (SiMe); exact masscalculated for C₄₀H₇₀O₅Si₂ [M]⁺ 686.4762, found 686.4789.

(23S)-25-Dehydro-2-Methylene-19-nor-1α-hydroxyvitamin D₃-26,23-lactone(HLV)

To a suspension of NaH (60% oil dispersion, 4 mg, 100 μmol) was added asolution of 10 (5 mg, 7 μmol) in THF (3 mL) at 0° C., and the mixturewas stirred at the same temperature for 30 minutes. To the mixture wasadded saturated aqueous NH₄Cl solution, and the aqueous layer wasextracted with Et₂O. The organic layer were combined and washed withsaturated aqueous NaCl solution, dried over MgSO₄, and concentrated. Theresidue was purified by flash column chromatography on silica gel (5%EtOAc/hexanes) to give protected vitamin 12 (4 mg, 6 μmol, 81%) as acolorless oil. ¹H NMR (CDCl₃, 900 MHz) δ 6.22 (dd, J=2.7, 2.7, 1H,=CH₂), 6.20 (d, J=11.3 Hz, 1H, 6-H), 5.82 (d, J=11.3 Hz, 1H, 7-H), 5.61(dd, J=2.7, 2.7 Hz, 1H, =CH₂), 4.96 (s, 1H, =CH₂), 4.90 (s, 1H, =CH₂),4.64 (m, 1H, CH—O—), 4.42 (m, 2H, 1β- and 3α-H), 3.06 (dddd, J=17.1,7.2, 2.7, 2.7 Hz, 1H) 2.81 (dm, J=12.6 Hz, 1H), 2.54 (dd, J=12.6, 5.4Hz, 1H), 2.52 (dddd, J=17.1, 5.4, 3.6, 2.7 Hz, 1H), 2.46 (dd, J=13.5,5.4 Hz, 1H), 2.28 (dd, J=13.5, 2.7 Hz, 1H), 2.16 (dd, J=12.6, 8.1 Hz,1H), 2.01 (dm, J=12.6 Hz, 1H), 1.99 (dd, J=12.6, 7.2 Hz, 1H), 1.01 (d,J=6.3 Hz, 3H), 0.89 (s, 9H, Si-t-Bu), 0.85 (s, 9H, Si-t-Bu), 0.55 (s,3H), 0.07, 0.05, 0.04, and 0.01 (each s, each 3H, 4× SiCH₃); ¹³C NMR(CDCl₃) δ 170.66, 153.18, 140.96, 135.05, 133.24, 122.51, 122.11,116.57, 106.44, 75.40, 72.82, 71.73, 57.07, 56.49, 47.88, 45.98, 43.73,40.84, 38.70, 34.72, 33.28, 28.90, 27.94, 26.05, 25.96, 23.57, 22.43,18.81, 18.47, 18.36, 12.31, −4.67 (3× SiMe), −4.88 (SiMe); exact masscalculated for C₃₉H₆₆O₄Si₂Na [M+Na]⁺ 677.4397, found 677.4407.

To a solution of 12 (3 mg, 5.3 μmol) in MeCN (1 mL) was added HF/MeCN(1:9, 1 mL) at 0° C., and the mixture was stirred at the sametemperature for 30 minutes. To the mixture was added saturated aqueousNaHCO₃ solution, and the aqueous layer was extracted with EtOAc. Theorganic layer was washed with saturated aqueous NaCl solution, driedover MgSO₄, and concentrated. The residue was purified by flash columnchromatography on silica gel (20% EtOAc/hexanes) to give HLV (2 mg, 4.6μmol, 88%) as a colorless oil. For analytical purpose a sample of thefinal product HLV was further purified by HPLC (Zorbax Eclipse XDB-C18,15% MeOH/H₂O, 3 mL/minute, Rv=21.8 mL); UV (in ethanol) λ_(max) 243,251, 261; ¹H NMR (CDCl₃, 800 MHz) δ 6.34 (d, J=11.2 Hz, 1H, 6-H), 6.23(dd, J=2.4, 2.4 Hz, 1H, =CH₂), 5.88 (d, J=11.2 Hz, 1H, 7-H), 5.62 (dd,J=2.4, 2.4 Hz, 1H, =CH₂), 5.11 (s, 1H, =CH₂), 5.09 (s, 1H, =CH₂), 4.65(m, 1H, —CH—O—), 4.49 (m, 1H), 4.46 (m, 1H), 3.07 (dddd, J=16.8, 8.0,2.4, 2.4 Hz, 1H) 2.87 (dd, J=13.6, 4.8 Hz, 1H), 2.81 (dm, J=12.8 Hz,1H), 2.57 (dd, J=13.6, 4.0 Hz, 1H), 2.53 (dddd, J=16.8, 5.6, 3.2, 3.2Hz, 1H), 2.33 (dd, 13.6, 5.6 Hz, 1H), 2.27 (dd, J=12.8, 8.0 Hz, 1H),2.01 (m, 2H), 1.02 (d, J=6.4 Hz, 3H), 0.57 (s, 3H); exact masscalculated for C₂₇H₃₈O₄Na [M+Na]⁺ 449.2668, found 449.2666.

(23R)-25-Dehydro-2-Methylene-19-nor-1α-hydroxyvitamin D₃-26,23-lactone(GC-3)

To a suspension of NaH (60% oil dispersion, 4 mg, 100 μmol) was added asolution of 11 (3 mg, 5.1 μmol) in THF (3 mL) at 0° C., and the mixturewas stirred at the same temperature for 30 minutes. To the mixture wasadded saturated aqueous NH₄Cl solution, and the aqueous layer wasextracted with Et₂O. The organic layer were combined and washed withsaturated aqueous NaCl solution, dried over MgSO₄, and concentrated. Theresidue was purified by flash column chromatography on silica gel (5%EtOAc/hexanes) to give protected vitamin 13 (1.5 mg, 2.3 μmol, 45%) as acolorless oil. ¹H NMR (CDCl₃, 900 MHz) δ 6.21 (dd, J=2.7, 2.7 Hz,1H,=CH₂), 6.20 (d, J=10.8 Hz, 1H, 6-H), 5.82 (d, J=10.8 Hz, 1H, 7-H),5.61 (dd, J=2.7, 2.7 Hz, 1H, =CH₂), 4.96 (s, 1H, =CH₂), 4.91 (s, 1H,=CH₂), 4.59 (ddt, J=14.4, 14.4, 7.2 Hz, 1H, —CH—O—), 4.41 (m, 2H, 1β-and 3α-H), 3.04 (dddd, J=16.7, 7.2, 2.7, 1.8 Hz, 1H) 2.81 (dm, J=12.6Hz, 1H), 2.54 (dddd, J=16.7, 6.3, 3.6, 2.7 Hz, 1H), 2.49 (dd, J=13.5,6.3 Hz, 1H), 2.45 (dd, J=13.5, 4.5 Hz1H), 2.32 (dd, J=13.5, 3.6 Hz, 1H),2.17 (dd, J=13.5, 9.0 Hz, 1H), 1.02 (d, J=7.2 Hz, 3H), 0.88 (s, 9H,Si-t-Bu), 0.85 (s, 9H, Si-t-Bu), 0.55 (s, 3H), 0.07, 0.05, 0.03, and0.01 (each s, each 3H, 4× SiCH₃); ¹³C NMR (CDCl₃) δ 170.57, 153.12,140.92, 134.94, 133.21, 122.52, 122.07, 116.55, 106.51, 72.71, 71.83,56.80, 56.35, 47.80, 45.87, 42.54, 40.72, 38.79, 34.27, 33.90, 28.87,28.24, 25.99 (2×-t-Bu-Si), 23.54, 22.38, 19.48, 18.45, 12.23, −4.66 (3×SiMe), −4.90 (SiMe); exact mass calculated for C₃₉H₆₆O₄Si₂Na [M+Na]⁺677.4397, found 677.4407.

To a solution of 13 (2 mg, 4 μmol) in MeCN (1 mL) was added HF/MeCN(1:9, 1 mL) at 0° C., and the mixture was stirred at the sametemperature for 30 minutes. To the mixture was added saturated aqueousNaHCO₃ solution, and the aqueous layer was extracted with EtOAc. Theorganic layer was washed with saturated aqueous NaCl solution, driedover MgSO₄, and concentrated. The residue was purified by flash columnchromatography on silica gel (20% EtOAc/hexanes) to give GC-3 (1 mg, 2μmol, 60%) as a colorless oil. For analytical purpose a sample of thefinal product GC-3 was further purified by HPLC (Zorbax Eclipse XDB-C18,15% MeOH/H₂O, 3 mL/minute, Rv=22.6 mL)); UV (in ethanol) λ_(max) 243,251, 261; ¹H NMR (CDCl₃, 800 MHz) δ 6.35 (d, J=11.2 Hz, 1H, 6-H), 6.22(dd, J=2.4, 2.4 Hz, 1H, =CH₂), 5.88 (d, J=11.2 Hz, 1H, 7-H), 5.62 (dd,J=2.4, 2.4 Hz, 1H, =CH₂), 5.12 (s, 1H, =CH₂), 5.09 (s, 1H, =CH₂), 4.59(ddt, J=14.4, 13.6, 7, 2 Hz, 1H, CH—O—), 4.49 (m, 1H), 4.47 (m, 1H),3.05 (dddd, J=16.8, 7.2, 2.4, 2.4 Hz, 1H) 2.84 (dd, J=13.6, 4.0 Hz, 1H),2.81 (dm, J=15.2 Hz, 1H), 2.58 (dd, J=13.6, 4.0 Hz, 1H), 2.55 (dddd,J=16.8, 6.4, 3.2, 3.2 Hz, 1H), 2.33 (dd, J=13.6, 6.4 Hz, 1H), 2.30 (dd,J=13.6, 8.8 Hz, 1H), 1.03 (d, J=6.4 Hz, 3H), 0.57 (s, 3H); exact masscalculated for C₂₇H₃₈O₄Na [M+Na]⁺ 449.2668, found 449.2688.

Biological Activity Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat vitamin D receptor was expressed in E. coliBL21(DE3) Codon Plus RIL cells and purified to homogeneity using twodifferent column chromatography systems. The first system was a nickelaffinity resin that utilizes the C-terminal histidine tag on thisprotein. The protein that was eluted from this resin was furtherpurified using ion exchange chromatography (S-Sepharose Fast Flow).Aliquots of the purified protein were quick frozen in liquid nitrogenand stored at −80° C. until use. For use in binding assays, the proteinwas diluted in TEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH 7.4, 5 mM DTT, 150 mMKCl) with 0.1% Chaps detergent. The receptor protein and ligandconcentration was optimized such that no more than 20% of the addedradiolabeled ligand was bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmol) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 mL of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocyiic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/mL. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In Vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24OHase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.RLU=relative luciferase units.

Antagonism was tested by adding a combination of 1,25(OH)₂D₃ and thecompound in the same well keeping the final ethanol concentration thesame.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (Suda et al.J. Nutr. 100:1049, 1970) (0.47% Ca) diet+vitamins AEK for one weekfollowed by Diet 11 (0.02% Ca)+AEK for 3 weeks. The rats were thenswitched to a diet containing 0.47% Ca for one week followed by twoweeks on a diet containing 0.02% Ca. Dose administration began duringthe last week on 0.02% calcium diet. Four consecutive ip doses weregiven approximately 24 hours apart. Twenty-four hours after the lastdose, blood was collected from the severed neck and the concentration ofserum calcium determined as a measure of bone calcium mobilization. Thefirst 10 cm of the intestine was also collected for intestinal calciumtransport analysis using the everted gut sac method. Antagonism wastested by administering a combination of 1,25(OH)₂D₃ and the compound tothe animal simultaneously.

Both GC-3 and HLV bind to the vitamin D receptor, but both of thesecompounds are less active in this respect than is 1α,25-dihydroxyvitamin D₃ (see FIG. 1). Both GC-3 and HLV also show lessactivity than 1,25-(OH)₂D₃ in inducing differentiation of HL-60 cells(FIG. 2). GC-3 shows antagonistic activity when administered along withthe native hormone (1α, 25-dihydroxyvitamin D₃) as shown in FIGS. 3 and5, and HLV shows antagonistic activity to an even greater extent asshown in FIGS. 4 and 5. GC-3 and HLV have no calcemic activity whenmeasured by bone calcium mobilization even when given at the dose of2,340 pmol/day (see FIG. 6). However, both GC-3 and HLV do retain theability to elevate intestinal calcium transport (FIG. 7). Because thesecompounds act as antagonists in vitro and weak agonists in vivo, thesecompounds could serve as useful therapeutic agents when administeredlocally to some tissues. These compounds may thus find use in therapiesfor treating asthma, hypercalcemia, eczema, sarcoidosis, and vitamin Dintoxication.

The compounds of the invention are useful in applications whereantagonization of the vitamin D receptor is desired. Such applicationsmay include the treatment of asthma or eczema in animal subjectssuffering from asthma or eczema. Therefore, in some embodiments, amethod of preventing or treating asthma or eczema in an animal subjectincludes administering to the animal subject, an effective amount of thecompound or compounds of the invention or a pharmaceutical compositionthat includes the compound or compounds. Administration of the compoundor compounds or the pharmaceutical composition to the subject inhibitsor reduces asthma or eczema in the animal subject.

For treatment purposes, the compounds defined by formulas 1A, 1B, 1A1,1B1, 1A2, and 1B2 may be formulated for pharmaceutical applications as asolution in innocuous solvents, or as an emulsion, suspension ordispersion in suitable solvents or carriers, or as pills, tablets orcapsules, together with solid carriers, according to conventionalmethods known in the art. Any such formulations may also contain otherpharmaceutically acceptable and non-toxic excipients such asstabilizers, anti-oxidants, binders, coloring agents or emulsifying ortaste-modifying agents. Pharmaceutically acceptable excipients andcarriers are generally known to those skilled in the art and are thusincluded in the instant invention. Such excipients and carriers aredescribed, for example, in “Remingtons Pharmaceutical Sciences” MackPub. Co., New Jersey (1991), which is hereby incorporated by referencein its entirety and for all purposes as if fully set forth herein.

The compounds may be administered orally, topically, parenterally,rectally, or transdermally. The compounds are advantageouslyadministered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentarycanal, or in the form of creams, ointments, patches, or similar vehiclessuitable for transdermal applications. In some embodiments, doses offrom 0.001 μg to about 1 mg per day of the compound are appropriate fortreatment purposes. In some such embodiments an appropriate andeffective dose may range from 0.01 μg to 1 mg per day of the compound.In other such embodiments an appropriate and effective dose may rangefrom 0.1 μg to 500 μg per day of the compound. Such doses will beadjusted according to the type of disease or condition to be treated,the severity of the disease or condition, and the response of thesubject as is well understood in the art. The compound may be suitablyadministered alone, or together with another active vitamin D compound.

Compositions for use in the invention include an effective amount ofGC-3 and/or HLV as the active ingredient or ingredients, and a suitablecarrier. An effective amount of the compound or compounds for use inaccordance with some embodiments of the invention will generally be adosage amount such as those described herein, and may be administeredtopically, transdermally, orally, nasally, rectally, or parenterally.

The compounds of formula 1A and formula 1B may be advantageouslyadministered in amounts sufficient to effect the differentiation ofpromyelocytes to normal macrophages. Dosages as described above aresuitable, it being understood that the amounts given are to be adjustedin accordance with the severity of the disease, and the condition andresponse of the subject as is well understood in the art. As noted, thecompounds of formula 1A and formula 1B may be present as a mixture ofthe two compounds. In some mixtures, the mixture may include thecompound of formula 1A and the compound of formula 1B. In someembodiments, the mixture includes the compound of formula 1A and thecompound of formula 1B, and the ratio of the compound of formula 1A tothe compound of formula 1B ranges from 50:50 to 99.9:0.1. In some suchembodiments, the ratio of the compound of formula 1A to the compound offormula 1B ranges from 70:30 to 99.9:0.1, from 80:20 to 99.9:0.1, from90:10 to 99.9:0.1, or from 95:5 to 99.9:0.1. In other embodiments, themixture includes the compound of formula 1A and the compound of formula1B, and the ratio of the compound of formula 1B to the compound offormula 1A ranges from 50:50 to 99.9:0.1. In some such embodiments, theratio of the compound of formula 1B to the compound of formula 1A rangesfrom 70:30 to 99.9:0.1, from 80:20 to 99.9:0.1, from 90:10 to 99.9:0.1,or from 95:5 to 99.9:0.1.

The compound or compounds may be formulated as creams, lotions,ointments, aerosols, suppositories, topical patches, pills, capsules ortablets, or in liquid form as solutions, emulsions, dispersions, orsuspensions in pharmaceutically innocuous and acceptable solvent oroils, and such preparations may contain, in addition, otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100 microns.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e., a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

All references cited herein are specifically incorporated by referencein their entireties and for all purposes as if fully set forth herein.

It is understood that the invention is not limited to the embodimentsset forth herein for illustration, but embraces all such forms thereofas come within the scope of the following claims.

1. A compound having the formula 1A, 1B, or a mixture thereof,

wherein X¹ and X² are independently selected from H and hydroxyprotecting groups.
 2. The compound of claim 1, wherein X¹ and X² areboth hydroxy protecting groups.
 3. The compound of claim 2, wherein X¹and X² are both t-butyldimethylsilyl groups.
 4. The compound of claim 1,wherein X¹ and X² are both H and the compound has the formula 1A1 or1B1:


5. A pharmaceutical composition, comprising an effective amount of thecompound of claim 4 and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition of claim 5 wherein the effective amountcomprises from about 0.01 μg to about 1 mg of the compound per gram ofthe composition.
 7. The pharmaceutical composition of claim 5 whereinthe effective amount comprises from about 0.1 μg to about 500 μg of thecompound per gram of the composition.
 8. The compound of claim 1,wherein X¹ and X² are both H and the compound has formula 1A2 or 1B2:


9. A method of treating a subject suffering from a biological disorder,comprising administering an effective amount of the compound or themixture of the compounds of claim 1 to the subject, wherein X¹ and X²are both H and the compound has the formula 1A1 or 1B1,


10. The method of claim 9, wherein the compound or mixture of compoundsantagonizes the vitamin D receptor in the subject after administration.11. The method of claim 9, wherein the biological disorder is asthma.12. The method of claim 9, wherein the biological disorder is eczema.13. The method of claim 9, wherein the compound, the mixture, or thepharmaceutical formulation is administered orally, parenterally,rectally, transdermally, or topically to the subject.
 14. The method ofclaim 9, wherein the compound, the mixture of compounds, or thepharmaceutical formulation is administered by delivering the compound,mixture of compounds or pharmaceutical formulation in an aerosol. 15.The method of claim 9, wherein the compound or the mixture of compoundsis administered in a dosage of from 0.01 μg per day to 1 mg per day. 16.The method of claim 9, wherein the compound has the formula 1A2 or 1B2,


17. A method of treating hypercalcemia, sarcoidosis, or vitamin Dintoxication in an animal subject suffering from hypercalcemia,sarcoidosis, or vitamin D intoxication, comprising administering aneffective amount of the compound, or the mixture of compounds, of claim1 to the animal subject, wherein X¹ and X² are both H and the compoundhas the formula 1A1 or 1B1,


18. The method of claim 17, wherein the compound, the mixture ofcompounds, or the pharmaceutical formulation is administered using aninhaler or nebulizer.
 19. The method of claim 17, wherein the compoundis administered orally, parenterally, rectally, transdermally, ortopically to the subject.
 20. The method of claim 17, wherein thecompound has the formula 1A2 or 1B2,


21. The compound of claim 1, wherein the mixture thereof comprises thecompound of formula 1A and the compound of formula 1B, and the ratio ofthe compound of formula 1A to the compound of formula 1B ranges from50:50 to 99.9:0.1.
 22. The compound of claim 21, wherein the ratio ofthe compound of formula 1A to the compound of formula 1B ranges from70:30 to 99.9:0.1.
 23. The compound of claim 1, wherein the mixturethereof comprises the compound of formula 1A and the compound of formula1B, and the ratio of the compound of formula 1B to the compound offormula 1A ranges from 50:50 to 99.9:0.1.
 24. The compound of claim 23,wherein the ratio of the compound of formula 1B to the compound offormula 1A ranges from 70:30 to 99.9:0.1.